The invention relates generally to the modification of cytotoxic T cells by treatment with a selected cytokine, and to the use of such modified cells in cancer therapy.
The human T cell line TALL-104 (CD3/TCRxcex1xcex2*CD8*CD16xe2x88x92) [A. Cesano et al, In Vitro Cell. Dev. Biol., 28A:648 (1992); A. Cesano et al, J. Immunol., 151:2943-2957 (1993); and A. Cesano et al, Cancer Immunol, Immunoth., 40:139 (1995)] is endowed with MHC non-restricted killer activity and has been reported as useful, when lethally irradiated, against a broad range of tumors across several species, while sparing cells from normal tissues. As taught by the inventors"" prior publications and patents cited above, unmodified TALL-104 cells are available from the American Type Culture Collection, 10801 University Boulevard Manassas, Va. 20110-2209 under Accession Number CRL 11386 and are described in U.S. Pat. No. 5,272,082. These cells may be preferably modified by lethal xcex3-irradiation and/or by stimulation in the cytokine interleukin 2 (IL-2) or Interleukin 12 (IL-12) to provide them with an increased cytotoxicity against tumor and virus-infected targets.
Such modification methods have been described in detail in International Patent Publication No. WO94/26284, published Nov. 24, 1994, which is incorporated by reference herein. For example, one modification step includes in vitro treatment of the TALL-104 cells with one or both of the two interleukins, recombinant human (rh) IL-2 and rhIL-12. When used independently to treat the cell line, IL-2 and IL-12 can induce the cell line""s cytotoxic activity. When these cytokines are used together to modify the cell line, the modified cell line displays additive or increased cytotoxic effects. This results in a significant increase in cytotoxic activity and recycling capability, ultimately leading to 100% elimination of tumor targets at an E:T ratio less than 0.1:1 [Cesano et al, J. Immunol., 151:2943 (1993)].
Another known modification step involves the exposure of the TALL-104 cell line to lethal irradiation to confer irreversible loss of growth capability with full retention of cytotoxic activity, both in vitro and in vivo. This is achieved by subjecting the cell line to y-irradiation just prior to its use. Preferably, the cells are irradiated at 4000 rads using a 137Cs source. As described in International Patent Publication No. WO94/26284, irradiation of TALL-104 cells provides a modified cytotoxic cell line that has lost its proliferative ability and, therefore, the possibility of growing in an unrestrained fashion in the rag, recipient organism. These modified TALL-104 cells have been used in methods for the treatment of various cancers in humans and animals. See, also, U.S. Pat. Nos. 5,683,690; 5,702,702 and 5,820,856, and International patent publication No. WO98/48630, all incorporated herein by reference.
Other cytotoxic cells have also been described, such as the TALL-103/2 cells. See, U.S. Pat. No. 5,272,082 and A. Cesano et al, J. Immunol, 151:2943-2957 (1993); S. Visonneau et al, Cell Immunol., 165:252-265 (1995); and A. Cesano et al, J. Immunol., 160:1106-1115 (1998). However, TALL-103/2 cells, stimulated with IL-2 or IL-12, have been noted to have a limited spectrum of tumor target reactivities and display low levels of killing. These cells do not grow in severe combined immuno-deficient (SCID) mice. Thus, at present, TALL-103/2 cells have not appeared promising for clinical use.
Among the known cytokines, Interleukin-15 (IL-15) is a relatively novel T cell growth factor that shares some activities and receptor components with IL-2 [U.S. Pat. No. 5,747,024; J. G. Giri et al, J. Leuko. Biol., 57(5):763-6 (May 1995); L. S. Quinn et al, Endocrinol., 136(8):3669-72 (August 1995)]. IL-15 utilizes the xcex2 and xcex3 chains of the IL-2 receptor for signal transduction, but uses a different subunit (xcex1) to bind to the cells. The expression pattern of IL-15 xcex1 receptor is distinct from that of IL-2 xcex1 receptor. IL-15 has been shown to induce LAK cell functions in vitro at high doses of about 100 ng/ml by a CD18-dependent, perforin-related mechanism [A. M. Gamero et al, Cancer Res., 55(21):4988-94 (November 1995)]. IL-15 is produced by monocytes and dendritic cells and has been shown to induce cytokine production in human T helper cells, and adhesion receptor redistribution in T lymphocytes. It has been described to stimulate proliferation of xcex3xcex4 T cells and act synergistically with other stimuli in inducing lymphokine production thereby [See, also, W. E. Carson et al, J. Clin. Invest. 96(6):2578-82 (December 1995); H. Jonuleit et al, J. Immunol., 158(6):2610-5 (Mar. 15, 1997); V. E. Garcia et al, J. Immunol., 160(9):4322-9 (May 1998); A. Mori et al, J. Immunol 156(7):2400-5 (April 1996); M. Nieto et al, Euro. J. Immunol., 26(6):1302-7 (June 1996); M. K. Kennedy et al, J. Clin. Immunol., 16(3):134-43 (May 1996)]. IL-15 has also been described as a vaccine adjuvant [U.S. Pat. No. 5,747,0241], a therapeutic [U.S. Pat. No. 5,660,824], and an inducer of angiogenesis [A. L. Angiolillo et al, Biochem. Biophys. Res. Comm., 2(1):231-7 (Apr. 7, 1997)]. IL-15 has been said to have IL-2-like stimulating activities on T lymphocytes and NK cells [P. Allavena et al, J. Leuko. Biol., 61(6):729-35 (June 1997); J. P. DiSanto, Current Biol., 7(7):R424-6 (Jul. 1, 1997); R. Evans et al., Cell. Immunol., 179(1):66-73 (Jul. 10, 1997)].
There exists a need in the art for methods for further enhancing the characteristics of cytotoxic T cells useful for therapy.
In one aspect, the invention provides a method of modifying, or reversibly modifying, the phenotype and function of cytotoxic T cells while retaining the cytotoxicity of the cells comprising the steps of:
(a) culturing said cells in an effective amount of IL-15 thereby obtaining a high yield of a cell having a first phenotype;
(b) culturing the IL-15 stimulated cells in an effective amount of IL-2, thereby altering the first phenotype to a second phenotype; and
(c) optionally repeating steps (a) and (b) a selected number of times.
In another aspect, the invention provides a method of modifying a cytotoxic T cells while retaining the cytotoxicity of the cells comprising the steps of:
(a) culturing said cells in an effective amount of IL-2, thereby obtaining a first modified cell;
(b) culturing the IL-2 stimulated cells in an effective amount of IL-15; thereby obtaining a second modified cell; and
(c) optionally repeating steps (a) and (b) a selected number of times.
The first and second modified cells from either method above demonstrate a change in at least one characteristic, such as increased proliferation, differentiation, growth, phenotype, adhesion molecule expression, biodistribution, cytokine production profile, level of cytotoxic activity, and tumor target spectrum. Desirably the cells are TALL-104 cells or TALL-103/2 cells.
In one embodiment of the first method, TALL-104 cells are cultured in an effective amount of IL-15, wherein said cells grow at a rate faster than when stimulated by IL-2, and have an altered phenotypic profile; and then the IL-15 stimulated TALL-104 cells are cultured in an effective amount of IL-2. In an embodiment of the second embodiment, the modification of cell characteristics is accomplished by first culturing TALL-104 cells in an effective amount of IL-2 and then culturing the IL-2 stimulated TALL-104 cells in an effective amount of IL-15.
In yet another aspect, the invention provides a method of modifying TALL-104 cells comprising culturing TALL-104 cells in an effective amount of IL-15, wherein said cells grow at a rate faster than when stimulated by IL-2, and have an altered phenotypic, cytotoxic and cytokine profile. The modified cells have an increased level of cytotoxicity or another change in a characteristic such as increased proliferation, differentiation, growth, phenotype, adhesion molecule expression, biodistribution, cytokine production profile, and tumor target spectrum. In one embodiment of this method the cytokine profile includes increased expression of IL-10, GM-CSF, TNF-xcex1 and TNF-5 and decreased expression of gamma interferon (IFN-xcex3) by the modified TALL-104 cells. In another embodiment, the modified phenotype includes increased expression of the cytotoxic adhesion/activation marker CD56 and/or decreased expression of the adhesion molecule CD38.
In still another aspect, the invention provides a method for increasing the levels of cytotoxic activity and spectrum of tumor target recognition of TALL-103/2 cells comprising culturing TALL-103/2 cells in an effective amount of IL-15, wherein said cells grow at a faster rate and have an expanded tumor target spectrum of cytotoxicity than when stimulated by IL-2.
In yet a further aspect, the invention provides modified TALL-104 cells, which are produced by stimulating said cells in an effective amount of IL-15.
In another aspect, the invention provides modified TALL-103/2 cells having an increased cytotoxicity, which are produced by stimulating said cells in an effective amount of IL-15.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.